Filoviruses have been associated with episodic, but increasingly frequent outbreaks of a highly lethal hemorrhagic fever, and are agents of concern for bioterrorism. Unfortunately, our grasp of the natural ecology of these viruses and the variables that control their emergence and virulence remains limited, as does our ability to treat filovirus infections. This is partly the result of a crucial knowledge gap: we have only a fragmentary understanding of the virus-host molecular interactions that are required for infection and that control the susceptibility of cells from different hosts. As a case in point, essential filovirus receptor(s) have remained elusive for a decade or more, despite considerable efforts to isolate them. However, we very recently showed that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is a critical cellular receptor for filovirus entry, infection, and pathogenesis. The overall goals of this proposal are to define the molecular mechanism by which NPC1 mediates filovirus entry and infection, and to investigate the implications of this newly discovered virus-receptor interaction for filovirus host range. To fulfill these objectives, he PI has assembled a multidisciplinary team that includes two leading structural biologists of viruses, an organization dedicated to surveillance of emerging pathogens in a unique network of animal sampling sites in filovirus-endemic central Africa, and a virologist who will corroborate findings from surrogate viruses with authentic filoviruses under BSL-4 containment. Together, we will: (1) Employ peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to define interaction surfaces between GP and NPC1 and map binding-induced conformational changes; (2) Identify sequences in GP and NPC1 that comprise the filovirus GP-NPC1 binding interfaces and influence post-binding steps in filovirus entry; (3) Determine the X-ray crystal structure of a GP-NPC1 complex; (4) Uncover the influence of GP- NPC1 interaction on filovirus host cell range; and (5) Determine the mechanistic role of GP- NPC1 interaction in filovirus entry.